Project Summary/Abstract Embryonic stem cells (ESCs) form the basis for transformative cell therapies for retinal blindness, which affects over 300M worldwide. Yet, the mechanisms by which ubiquitous chromatin modifiers, like WDR5, cooperate with broadly expressed, embryonic transcription factors (TFs), like p53 and MAX, control retinogenesis are unknown. This knowledge gap affects critical fields. p53 activation, a feature of CHARGE and other syndromes, triggers retinal defects via undetermined pathways. Transplantation of p53-mutant ESC-derived retinal cells continue in clinical trials but it is not known if p53 regulates ESC retinal fate determination. A $1 billion effort to develop WDR5 inhibitors is ongoing. Yet, little is known about WDR5 beyond its role in promoting transcription as a co- factor of the MLL chromatin modifying complex, which methylates lysine 4 on histone H3 (H3K4me). Thus, predicting the outcome of these medical interventions remain challenging. During the PI?s K08 award period, we discovered that WDR5 regulates p53 stability to promote retinogenesis. Further, our preliminary data reveals that WDR5 directly interacts with p53 and MAX to regulate non-retinal lineage specification, mesoderm and germ cell/meiosis-related transcription. The objective of the proposed research is to understand how interplay of ubiquitous chromatin modifiers and TFs at a critical developmental window trigger the earliest events of retinogenesis. This proposal tests the central hypothesis that WDR5 interacts with p53 and MAX on chromatin in a time-dependent manner to promote retinogenesis by activating retinal-specific genes and by repressing non- retinal, lineage-specifying loci. We will test this hypothesis through three aims: (1) Delineate functions of WDR5, p53, and of loci that co-recruit WDR5 and p53, during retinogenesis; (2) Define molecular interactions of WDR5 and MAX that inhibit non-retinal fates during retinal specification; (3) Determine the role of the WDR5-p53 cell fate pathway during lineage specification of pluripotent cells in vivo. Our approach is significant and innovative because it employs state-of-the-art technologies, such as CUT&RUN, CRISPR-Cas9 editing, single cell transcriptome profiling, and ESC-derived 3D organoid platforms, to obtain foundational insights about the earliest events of retinogenesis. Our research will address non-canonical functions of popularly-studied proteins, such as roles for WDR5 that control eye field TF activity, trigger gene repression and cell fate functions of p53 and MAX distinct from tumorigenesis. Thus, gleaned insights will represent substantial departures from conventional views. Our insights will vertically advance and fundamentally alter our understanding of how ubiquitous chromatin modifiers and TFs interact in a temporal manner to initiate retinogenesis. Results from our studies will advance key concepts related to how p53 activation triggers retinal defects in p53-associated syndromes, mechanisms by which existing p53 alterations in ESC lines alter non-retinal lineage differentiation in ongoing cell therapy trials, and prediction of off-target effects of ?therapeutic? WDR5 inhibitors that are currently in development.